The ubiquitin-proteasome system (UPS) regulates the activity, localization and stability of thousands of proteins in the cell. By catalyzing the covalent attachment of ubiquitin to target proteins, the enzymes of the UPS regulate virtually every cellular process, including cell signaling, cell survival and cell division. The widespread influence of the UPS on biology also has important consequences for human health, as mutations in components of the UPS cause diseases such as cancer. Encouragingly, the vast number of enzymes and protein-protein interactions in the UPS are also providing new drug targets to treat disease. For these reasons, mechanistic studies of the UPS have the potential to reveal new insights into biological regulation and may also help us understand mechanisms of disease pathogenesis and therapy. A major focus of this proposal is to study the Anaphase-Promoting Complex/Cyclosome (APC). The APC is a multiprotein complex required for the attachment of ubiquitin to key proteins that regulate cell division. In the absence of APC activity, cells arrest in mitosis. Because the APC is essential for cell division, it may represent a useful drug target for the treatment of cancer. For these reasons, our lab has pioneered the development of small molecule inhibitors of the APC, called TAME and apcin. We have defined the mechanisms by which these inhibitors block APC activity. Here we propose to use these compounds to learn more about APC function, by identifying novel proteins that fail to be degraded in the presence of the inhibitors. In addition we plan to identify genes that influence the sensitivity of cells to APC inhibitors. Together these experiments may reveal new functions of the APC, and may help identify mutations in cancer cells that make them particularly sensitive to APC inhibition. The APC is regulated through a number of complex mechanisms that are not fully understood. Understanding APC regulation is important because proper timing of APC activation is essential for accurate chromosome segregation. Furthermore, we do not understand how the cell controls the level of expression of different APC subunits. To address these questions we plan to reconstitute key steps in APC-dependent proteolysis using purified components. We also plan to quantitatively determine the composition of the APC and associated proteins in different contexts to better understand its composition and function. Finally, we have developed a new approach to identify novel substrates of the ubiquitin-proteasome system. We propose to characterize these proteins to understand how they are degraded. We are particularly interested in identifying enzymes that remove ubiquitin from these substrates, thereby opposing their degradation. Together these studies will identify new pathways that regulate protein stability in the cell, and may provide new starting points for the development of drugs to treat disease.